Part:BBa_K1585300:Experience

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Applications of BBa_K1585300

The construct was confirmed by sequencing. Moreover, the expression of glgA was tested by an SDS-PAGE. In the picture below, you can see that glgA is expressed in strains containing BBa_K1585310 in a pSB1K30 expression vector.

SDS-PAGE of GlgA in comparison to mRFP After induction with IPTG at OD=0.6, glgA was expressed. Samples were taken 6 and 18.5 hours after induction. The small arrows indicates the expected bands for GlgA. mRFP in pSB1K30 (T7 promoter) was used as the negative control.

On top of that, the function of the construct was proven by an iodine staining with BL21 Gold (DE3) and BL21 Gold (DE3) expressing glgA in pSB1K30. The iodine staining is performed with Lugol's iodine which dyes glycogen resulting in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the brown color of BL21 Gold (DE3) expressing glgA indicates that the enzyme has the expected activity.

Iodine staining of BL21 Gold (DE3) with glgA in pSB1K30 compared to BL21 Gold (DE3) wild type Iodine staining was performed with overnight cultures which were adjusted to the same OD. The darker color of the strain expressing glgA shows that more glycogen is present. Therefore, the functionality of the glycogen synthase is shown.

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